The goal of this project is to develop quantitative genetic approaches for the identification of loci underlying virulence-associated traits in Candida albicans. C. albicans is the primary cause of oropharyngeal candidiasis (OPC) and esophageal candidiasis (OEC), conditions that continue to afflict HIV-infected patients even in the era of antiretroviral therapy. C. albicans is also a prevalent cause of life-threatening systemic infections, particularly in nosocomial and immunocompromised populations. However, the molecular factors that contribute to pathogenesis remain poorly defined, in part because of a lack of unbiased, genome-wide methods to investigate this species. In particular, the lack of a conventional sexual cycle has restricted the use of genome-wide association studies to study the mechanisms underlying commensalism and pathogenicity. To address the lack of forward genetic tools in C. albicans, we will develop Quantitative Trait Loci (QTL) mapping as a novel approach for the identification of genes contributing to pathogenesis in this species. This technique will utilize the specialized parasexual mating cycle in C. albicans, in which mated cells undergo a reduction in ploidy by a non-meiotic mechanism, for the generation of recombinant progeny. In addition, the advent of high-throughput genomic methods such as restriction site associated DNA sequencing (RAD-Seq) allows for rapid and inexpensive genotyping of recombinant progeny. As such, the proposed methodologies will fill the void for an unbiased genetic approach to define the factors promoting colonization, infection, and drug resistance in C. albicans. We present extensive preliminary data establishing the feasibility of QTL mapping in C. albicans. Each of the steps in the parasexual mating cycle can be achieved with high efficiency and a pipeline is in place for genomic analysis of recombinant parasexual progeny by RAD-Seq. In addition, clinical isolates have been identified that exhibit significant differences i important phenotypic traits such as epithelial cell damage and drug resistance. Genome sequencing of these isolates has revealed polymorphisms that likely contribute to inter-strain variation. In this application we will use QTL mapping to address (1) differences in the ability of strains to cause epithelial cell colonization and cell damage, and (2) mechanisms of resistance to the common antifungal drug fluconazole. Completion of these Aims will establish that quantitative genetic approaches can be exploited for the identification of novel factors contributing to pathogenesis in C. albicans. Furthermore, they will set the stage for future studies using QTL mapping as an unbiased and powerful approach to determine genetic factors underlying multiple aspects of pathogenicity in Candida species.